A spectrophotometer or colorimeter makes use of the
transmission of light through a solution to determine the
concentration of a solute within the solution. A spectrophtometer
differs from a colorimeter in the manner in which light is
separated into its component wavelengths. A spectrophotometer
uses a prism to separate light and a colorimeter uses filters.

Both are based on a simple design of passing light of a
known wavelength through a sample and measuring the amount of
light energy that is transmitted. This is accomplished by placing
a photocell on the other side of the sample. All molecules absorb
radiant energy at one wavelength of another. Those that absorb
energy from within the visible spectrum are known as pigments.
Proteins and nucleic acids absorb light in the ultraviolet range.
The following figure demonstrates the radiant energy spectrum
with an indication of molecules which absorb in various regions
of that spectrum.

The design of the single beam spectrophotometer involves a
light source, a prism, a sample holder and a photocell. Connected
to each are the appropriate electrical or mechanical systems to
control the illuminating intensity, the wavelength, and for
conversion of energy received at the photocell into a voltage
fluctuation. The voltage fluctuation is then displayed on a meter
scale, is displayed digitally, or is recorded via connection to a
computer for later investigation.

Spectrophotometers are useful because of the relation of
intensity of color in a sample and its relation to the amount of
solute within the sample. For example, if you use a solution of
red food coloring in water, and measure the amount of blue light
absorbed when it passes through the solution, a measureable
voltage fluctuation can be induced in a photocell on the opposite
side. If now the solution of red dye is diluted in half by the
addition of water, the color will be approximately 1/2 as intense
and the voltage generated on the photocell will be approximately
half as great. Thus, there is a relationship between the voltage
and the amount of dye in the sample.

Given the geometry of a spectrophotometer, what is actually
measured at the photocell is the amount of light energy which
arrives at the cell. The voltage meter is reading the amount of
light TRANSMITTED to the photocell. Light transmission is not a
linear function, but is rather an exponential function. That is
why the solution was APPROXIMATELY half as intense when viewed in
its diluted form.

We can however monitor the transmission level and convert it
to a percentage of the amount transmitted when no dye is present.
Thus, if 1/2 the light is transmitted, we can say that the
solution has a 50% Transmittance. Note that it is always relative
to a solution containing no dye.

Transmittance is the relative percent of light passed
through the sample.

What makes all of this easy to use, however, is the
conversion of that information from a percent transmittance to an
inverse log function known as the Absorbance (or Optical
Density).

The Beer-Lambert Law

Definiton

Absorbance: The negative log of the transmittance.

A = - logT EQUATION G.1

This value is more useful in spectrophotometry than
transmittance, because of plot of absorbance vs concentration
yields a straight line. A plot of transmittance vs concentration
is an exponential. The - log calculates the inverse of
transmittance, so that absorbance increases with increasing
concentration. Transmittance would decrease as we increased the
amount of red dye in our example. The relationship of Absorbance
to concentration was shown by two biochemists to follow the
equation for a straight line, y = mx +b, where m is the slope of
the line and b is the y intercept. If the measurement is made in
such a way that b = 0 (that is, a solution containing no dye has
no absorbance), and if we substitute Absorbance for y,
concentration for x, and variant for m, we arrive at the
formulation of the Beer-Lambert Law:

Note that variant is equal to the slope of the straight line
which will result from a plot of absorbance (y axis) vs
concentration (x axis).

To use a spectrophotometer it is necessary to establish a
known series of dilutions containing known quantities of a
solute. One of these will contain no solute and is known as the
blank . It is used to adjust the instrument to read 100%
transmittance or 0 absorbance. In use, a 0% transmittance value
(infinite absorbance) is established by placing a curtain between
the light source and the photocell. Electronic control is then
exerted so that the meter will read 0% Transmittance on its
scale. The blank sample (containing no solute or dye) is
inserted, the curtain opened and the meter readjusted to read
100% transmittance. All other measures are then made by merely
inserting the samples into the light path and measuring the %
transmittance. Most spectrophotometers have a built in means of
direct conversion of this reading to absorbance.

After recording the absorbance for a series of standards, a
plot is made of the absorbance value (y axis) vs the
concentration (x axis). The slope of the line is the extinction
coefficient.

Note that this may be computed directly by rearrangement of
the Beer-Lambert law to

= A/C EQUATION G.2

This value can be calculated for each reading and the
average taken as the value of variant. Remember that this value
is a constant. Thus, once calculated, it can subsequently be used
to determine an unknown concentration by one more rearrangement
of the Beer-Lambert law

C = A/ EQUATION G.3

Any measured value of A can be readily converted to a
corresponding concentration merely by dividing the absorbance by .

The use of the Beer-Lambert law is easy to visualize with
red food coloring. It is not as easy to visualize, but none the
less, just as accurate to measure wavelengths of light which are
not visible. Either infra red or ultraviolet can be used. UV is
more useful to biologists since many molecules (all proteins and
nucleic acids) absorb ultraviolet light. The only changes that
need to be made is the use of quartz cuvettes instead of glass
tubes. Glass absorbs UV light and thus is inappropriate for use
in a UV spectrophotometer. An instrument capable of using visible
light (usually with a tungsten or halogen lamp source) and UV
light is known as a UV/Vis Spectrophotometer.

OPERATION OF B&L SPEC. 20

The most commonly encountered spectrophotometer is one
manufactured by Bausch and Lomb and known as the Spec 20. The 20
refers to the band size of light that it is capable of producing.
If the instrument is adjust to a wavelength of 730, for example,
it actually transmits light from 720 nm to 740 nm. Thus, it is
not as precise or refined as instruments designed for research
purposes where the wavelength may be controlled to a fraction of
a nanometer. It is, however, the standard workhorse instrument
found in nearly every lab.

Turn on the spectrophotometer and allow 10 minutes for warm
up of the instrument before use.

Adjust the wavelength to that specificied for the procedure
you are using.

Be sure the cover is closed on the cuvette holder and use
the left knob on the front panel to adjust the dark current such
that the meter is reading 0 transmittance. At this point, you are
simply adjusting the internal electronics of the instrument to
blank out any residual currents. This adjusts the lower limit of
measurements. It establishes that no light is equivalent to 0
transmittance or infinite absorbance.

Insert a clean cuvette containing the blank into the holder.
Be sure that the tube is clean, free of finger prints and that
the painted line marker on the tube is aligned with the mark on
the tube holder. Close the top of the tube holder. The blank for
this exercise is the solution containing no dopachrome, but all
other chemicals. The amount of solution placed in the cuvette is
not important, but is usually about 5 ml. It should approximately
reach the bottom of the logo printed on the side of the cuvette.

Adjust the meter to read 100% transmittance, using the right
knob on the front of the instrument. This adjusts the instrument
to read the upper limit of the measurements and establishes that
your blank will give a reading of 100% transmittance (0
absorbance).

Remove the blank from the instrument and recheck that your 0
transmittance value has not changed. If it does, wait a few
minutes for the instrument to stabilize and redo steps 1-5.
Periodically throughout the exercise, check that calibration of
the instrument is stable by re-inserting the blank and checking
that the 0 and 100% T values are maintained.

To read a sample, simply insert a cuvette holding your test
solution and close the cover. Read the transmittance value
directly on the scale.

Record the % transmittance of your solution, remove the test
tube cuvette and continue to read and record any other solutions
you may have.

It is possible to read the absorbance directly, but with an
analog meter (as opposed to a digital read out), absorbance
estimations are less accurate and more difficult than reading
transmittance. Absorbance can be easily calculated from the
transmittance value. Be sure that you note which value you
measure!

ABSORPTION SPECTRUM:

Analysis of pigments often requires a slightly different use
of a spectrophotometer. In the use of the instrument for
determination of concentration (Beer-Lambert Law), the wavelength
was pre-set and left at a single value throughout the use of the
instrument. This value is often given by the procedure being
employed, but can be determined by an analysis of the absorption
of a solution as the wavelength is varied.

The easiest means of accomplishing this is to use either a
dual beam spectrophotometer or a computer controlled instrument.
In either event, the baseline must be continously re-read as the
wavelength is altered.

To use a single beam spectrophotometer (such as the Spec
20), the machine is zeroed first, the wavelength is set, the
blank is adjusted and then the sample is inserted and read. The
wavelength is then adjusted up or down by some determined
interval, the zero is checked, the blank re-inserted and
adjusted, and the sample re-inserted and read. This procedure
continues until all wavelengths to be scanned have been read.

In this procedure, the sample remains the same, but the
wavelength is adjusted. Compounds have differing absorbtion
coefficients for each wavelength. Thus, each time the wavelength
is altered, the instrument must be recalibrated.

A dual beam spectrophotometer divides the light into two
paths. One beam is used to pass through a blank, while the
remaining beam passes through the sample. Thus, the machine can
monitor the difference between the two as the wavelength is
altered. These instruments usually come with a motor driven
mechanism for altering the wavelength, or scanning the sample.

The newer version of this procedure is the use of an
instrument which scans a blank, and places the digitalized
information in its computer memory. It then rescans a sample and
compares the information from the sample scan to the information
obtained from the blank scan. Since the information is
digitalized (as opposed to an analog meter reading), manipulation
of the data is possible. These instruments usually have direct
ports for connection to personal computers, and often have built
in temperature controls as well. This latter option would allow
measurement of hanges in absorbtion due to temperature changes
(known as hyperchromicity). These in turn can be used to monitor
viscosity changes, which is related to the degree of molecular
polymerization with the sample. For instruments with this
capability, the voltage meter scale has given way to a CRT
display, complete with graphics and built in functions for
statistical analysis.

A temperature controlled UV spectrophotometer capable of
reading several samples at pre-programmed time intervals is
invaluable for enzyme kinetic analysis. An example of this type
of instrument is the Beckman DU-70.

SPECIFIC PROCEDURES:

For routine use, substances to be monitored by
spectrophotometry are often reacted with dyes to form a complex
that is of another color, usually one easily read within the
visible light range, and with precision by an instrument such as
the Spec 20.